人血清中A、C、Y、W135群脑膜炎球菌荚膜多糖IgG抗体含量ELISA检测方法的建立  

Development of ELISA method for IgG content specific to meningococcal capsular polysaccharide of groups A,C,Y and W135

作　　者：王欣茹[1] 赵志强[1] 李亚南[2] 刘方蕾[1] 于旭博 孔素娟[1] 范锋锋[1] 林纪胜[3] 叶强[2] 谢贵林 

机构地区：[1]兰州生物制品研究所有限责任公司甘肃省疫苗工程技术研究中心,甘肃兰州730046 [2]中国食品药品检定研究院,北京100050 [3]美国阿拉巴马大学伯明翰分校病理学系呼吸道病原参考实验室,伯明翰35294 [4]中国生物技术股份有限公司,北京100029

出　　处：《中国生物制品学杂志》2014年第11期1448-1453,共6页Chinese Journal of Biologicals

基　　金：国家科技支撑计划课题(2008BAI66B01);国家"重大新药创制"专项(2013ZX09402-302-215);甘肃省科技重大专项(0801NKDA003);甘肃省"十二五陇药产业发展"专项

摘　　要：目的建立人血清中抗A、C、Y、W135群脑膜炎球菌荚膜多糖Ig G抗体含量的ELISA定量检测方法,并进行验证。方法筛选适用的酶标板,优化抗原包被浓度,确定ELISA检测过程的关键条件,对建立的ELISA方法的特异性、线性、准确性、精密度、最低检测值和稳定性进行验证,并建立实验室质控血清的质控检测范围。结果酶标板的板内CV为9.8%,板间CV为16.4%,符合检测使用要求。免疫血清经多糖吸收后,群特异性抗体含量与同群多糖吸收剂量呈明显的剂量依赖关系。12份美国CDC质控血清中各群Ig G抗体含量与血清的稀释度呈负相关,相关系数（r）和斜率（slope）均接近-1,线性关系良好;检测结果与CDC公布数据一致性良好,一致性相关系数A、C、W135、Y群分别为0.912 5、0.939 6、0.963 7和0.920 6,准确度较好。实验室内部质控血清Men20中各群Ig G抗体含量试验内和试验间精密度分别小于10%和20%,精密度较好;各血清群别的CV值均〈30%,稳定性较好;A、C、Y、W135群多糖Ig G抗体含量最低检测值分别为0.092、0.118、0.067和0.035μg/ml,质控血清的检测范围分别为：92.49~36.53、37.29~17.69、66.18~25.54和50.15~23.31μg/ml。结论建立了标准化的检测人血清中抗A、C、Y、W135群脑膜炎球菌多糖Ig G抗体含量的定量ELISA方法,并确定了实验室日常用质控血清Men20的质控检测值范围。Objective To develop and verify an ELISA method for quantitative determination of Ig G content specific to meningococcal capsular polysaccharide of groups A,C,Y and W135. Methods Appropriate ELISA plate was screened,while the antigen concentration for coating was optimized,and the key condition during determination by ELISA was determined. The developed ELISA method was verified for specificity,linearity,accuracy,precision,lowest limitation of quantitation（LLQ）and stability,based on which an in-house quality control range was established. Results The intraand inter-CVs of ELISA plate were 9. 8% and 16. 4% respectively,which met the requirements for determination. The group-specific antibody contents in immune sera after adsorption with polysaccharide were significantly dependent on the dosage of polysaccharide of the same group. The Ig G concentrations of various groups in 12 reference serum samples from CDC of USA were negatively related to the dilution,of which the correlation coefficient（r） and slope of determination curve were nearly to-1,indicating a good linearity. The determination result was consistent with the data published by the CDC. The correlation coefficients of consistencies of groups A,C,W135 and Y meningococcal capsular polysaccharide were 0. 912 5,0. 939 6,0. 963 7 and 0. 920 6,respectively,indicating a high accuracy. The intra- and inter-precisions of determination results of Ig G of various groups in in-house quality control serum sample Men20 were less than 10% and less than 20% respectively,indicating a high precision. The CVs of determination results of various groups were less than30%,indicating a high stability. The LLQs of Ig G concentrations specific to meningococcal capsular polysaccharide of groups A,C,Y and W135 were 0. 092,0. 118,0. 067 and 0. 035 μg / ml,while the quality control ranges were 92. 49 ~35. 53,37. 29 ~ 17. 69,66. 18 ~ 25. 54 and 50. 15 ~ 23. 31 μg / ml,respectively. Conclusion A standardized ELISA method for quantitative determination of Ig G content specifi

关 键 词：脑膜炎球菌 荚膜多糖 抗体 酶联免疫吸附试验 

分 类 号：R378.15[医药卫生—病原生物学] R392-33[医药卫生—基础医学]